Osteoclasts, cells of myeloid lineage, play a unique role in bone resorption, maintaining skeletal homeostasis in concert with boneproducing osteoblasts. Osteoclast development and maturation (osteoclastogenesis) is driven by receptor activator of NF-B ligand and macrophage-colony stimulating factor and invariably requires a signal initiated by immunoreceptor tyrosine-based activation motif (ITAM)-harboring Fc receptor common ␥ chain or DNAX-activating protein (DAP)12 (also referred to as KARAP or TYROBP) that associates with the cognate immunoreceptors. Here, we show that a third adaptor, YINM costimulatory motif-harboring DAP10, triggers osteoclastogenesis and bone remodeling. DAP10-deficient (DAP10 ؊/؊ ) mice become osteopetrotic with age, concomitant with a reduction in osteoclasts. The DAP10-associating receptor was identified as myeloid DAP12-associating lectin-1 (MDL-1), whose physiologic function has not been found. MDL-1-mediated stimulation of osteoclast precursor cells resulted in augmented osteoclastogenesis in vitro. MDL-1 associates with both DAP12 and DAP10 in osteoclasts and bone marrow-derived macrophages, where DAP10 association depends almost entirely on DAP12, suggesting a formation of MDL-1-DAP12/ DAP10 trimolecular complexes harboring ITAM/YINM stimulatory/ costimulatory motifs within a complex that could be a novel therapeutic target for skeletal and inflammatory diseases.immunoreceptor tyrosine-based activation motif-harboring adaptor ͉ osteoclast development ͉ synergistic signal

SummaryObjectiveThe anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl− current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model.DesignAdult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol.ResultsAt 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na+, K+ and Ca2+ currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance.ConclusionChanges in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.

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A 61-year-old man was referred to us for investigation of acute abdominal pain. Computed tomography showed a 5.9 × 5.3 × 5.0 cm lump of food residue in the jejunum, and a large amount of free air and ascites around the liver and right paracolic gutter. He underwent emergency laparotomy for suspected peritonitis from perforation by a foreign body in the small intestine. We identified purulent exudate in the abdominal cavity and perforation of a jejunal cystic mass, attached ~40 cm from Treitz's ligament at the anti-mesenteric side of the jejunum. Based on a diagnosis of jejunal duplication with perforation, we resected that part of the small intestine and performed intra-abdominal drainage. Pathological findings confirmed the diagnosis of a perforated gastrointestinal stromal tumor (GIST) in a true jejunal diverticulum. Histopathological evidence suggests that intestinal pressure and/or hemorrhage can cause perforation in the background of a true jejunal diverticulum. To our knowledge, this is the first case report of a perforated GIST in a true jejunal diverticulum.

We report a case of thigh emphysema resulting from perforated appendicitis. The patient was an 83-year-old man who had no apparent abdominal signs and was initially misdiagnosed as having psoas abscess. Magnetic resonance imaging of the pelvis revealed appendicitis, and a barium enema showed a leakage of enhanced contrast material from the appendix region down into the thigh. A retroperitoneal perforation of the retrocaecal appendix without peritonitis was diagnosed. The patient underwent an appendectomy and curettage of the retroperitoneal and psoas muscle spaces, as well as the thigh. He recovered gradually, though the abscess had extended into the hip joint and resulted in osteomyelitis, requiring an additional procedure of resection arthroplasty. The patient fully recovered with no signs of infection one year postoperatively.

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E (PGE) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE release and was attenuated by inhibition of either phospholipase A or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.

Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time-and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the nonionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p ¼ 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p ¼ 0.964). GTP-RhoA levels were significantly increased (p < 0.001), but Rho inhibitor-1 failed to suppress the membrane blebbing (p ¼ 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin-II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes.

Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.